This project has two parts, one investigating the communication between parasympathetic nerve development and SMG epithelial morphogenesis, and the second identifying mouse salivary gland stem/progenitor cell populations within the gland. The maintenance of progenitors as a reservoir of undifferentiated cells is required for organ development and regeneration. Parasympathetic nerves are a vital component of the progenitor niche during development. Injured adult organs do not regenerate after parasympathectomy, and there are few treatments to improve organ regeneration or prevent damage, particularly that caused by therapeutic irradiation. We used SMG explant culture and injured the tissue with irradiation. Restoring parasympathetic function in vitro with the neurotrophic factor neurturin, increases epithelial organ regeneration after irradiation. The progenitors survived, parasympathetic function was diminished, and epithelial apoptosis reduced expression of neurturin, which consequently increased neuronal apoptosis. Treatment with neurturin reduced neuronal apoptosis, restored parasympathetic function, and increased epithelial regeneration. We also analyzed irradiated human SMG biopsies and compared them with non-IR controls. NRTN was reduced by 60% after IR, but there was increased neuronal immunostaining due to increased sympathetic innervation. These data suggested that IR disrupts the balance of parasympathetic and sympathetic innervation in the salivary glands. We are using NRTN gene therapy to improve SMG function after IR. First, we investigated whether a NRTN-expressing adenovirus (NRTN-Adv) could be used to protect parasympathetic neurons and improve regeneration after IR. We used ex vivo fetal SMG culture to compare the NRTN-Adv to recombinant NRTN, showing they both improve gland growth after IR and reduce PSG apoptosis. Then, we used retro-ductal delivery of NRTN-Adv to mouse SMGs in vivo before IR. The mice were treated with increasing doses of NRTN-Adv or Control-Adv 24 hr before receiving a fractionated IR dose (6 Gy x 5 days). After 60 days, salivary flow was measured after pilocarpine (a secretogogue) stimulation. The Control-Adv treated glands had 50% reduction in salivary flow whereas, NRTN-Adv-treated glands had similar flow to non-IR glands. Further, markers of parasympathetic function, including vesicular acetylcholine transporter, decreased with IR but not with NRTN treatment. Our findings suggest that in vivo NRTN gene therapy prior to IR protects parasympathetic function and prevents IR-induced hypofunction. We continue to use viral vectors that express neurotrophic factors to infect salivary glands of mice and minipigs in vivo to improve the function of surviving nerves in repairing the gland. Our goal is to characterize cells that express keratin 5 (K5) and Keratin 14 (K14) in salivary glands to identify markers that could be used to isolate specific subpopulations and to understand the relationships among these cells. We use both mice that express transgenic reporters K5venus and K14rfp SMGs and single cell transcriptomic analysis of murine salivary development of wildtype mice. Our ongoing analysis will characterize the multiple cell populations and identify markers to isolate some of them by FACs. These cells may be useful to expand in vitro for use in regenerative therapy of irradiated salivary glands.